Allogeneic bone marrow transplantation (BMT) provides a potentially curative treatment for leukemias that are refractory to conventional therapy. In addition to providing hematopoietic rescue from myeloablative therapy, BMT offers an adoptive immunotherapy effect (graft-versus-leukemia-GvL) that can be beneficial in the elimination of residual leukemia. This was initially shown in cases where T cell depletion (TCD) has been used to prevent graft-versus-host disease (GvHD) but also experienced an increase in disease relapse (1). Indeed, relapse rates in high-risk patients (long-standing recurrent disease or relapse at the time of BMT) can be as high as 70% (2). Therefore, further improvement in disease-free survival is likely to depend on the antileukemic effectiveness of the transplant, i.e. maximizing the GvL effect.
Most experimental evidence suggests that GvL effectors are predominantly T cells that can either recognize allospecific molecules expressed on both normal and neoplastic hematopoietic cells or recognize cell surface molecules that are either unique to or preferentially expressed by the leukemia (3–7). Identification of specific cell populations that are important antileukemic effectors is an essential first step to successful GvL graft engineering and cellular immunotherapy.
Although several studies have suggested that γδ+ T cells may not be important primary effectors of GvHD (8–12), few have addressed the GvL potential of γδ+ T cells. Esslin (13) noted that in vitro activated γδ+ T cells can mediate broadly-based non-MHC restricted cytolytic activity to selected human tumor cell lines. Others have shown that γδ+ T cells can recognize unprocessed peptides, some of which are preferentially expressed on tumor cells (14–18). Finally, one report has shown cytotoxic anti-leukemic activity in a patient against B cell ALL by γδ+ T cells expressing the Vδ1 form of the T cell receptor (19). Taken together, these findings support a potential antileukemic role for γδ+ T cells.
Published data describing a series of 10 leukemia patients who developed an increased proportion of circulating CD3+CD4−CD8−Vδ1+γδ+ T cells between 60 and 270 days post-BMT from a partially mismatched related donor (PMRD) which continued for up to two years. Eight of these patients are surviving and remain free of disease, as compared to a DFS probability of 31% at 2.5 years among 100-day survivors with a normal number of γδ+ T cells (20). In addition, it has been recently shown that enrichment of the graft with γδ+ T cells may have contributed to the later development of increased γδ+ T cells (21). Regardless of the TCD protocol used, however, patients who developed increased γδ+ T cells showed the same cell phenotype and cytolytic function as well as a decreased incidence of relapse.
Allogeneic Bone Marrow Transplantation and Graft-Host Interactions: High-dose chemo/radiotherapy followed by bone marrow rescue provides a potentially curative treatment for a variety of leukemias and solid tumors that are refractory to conventional therapy. An alloreactive response, mediated by donor immunocompetent cells in the graft and directed against normal cells and tissues in the recipient can result in the development of graft-versus-host disease (GvHD). GvHD can occur in up to 50% of patients receiving unmodified, HLA-identical marrow, indicating that minor histocompatibility differences, not detected by conventional HLA matching techniques, can initiate this reaction (22, 23). For the majority of patients (approximately 70% ) who do not have matched sibling donor (MSD) alternative donors may be used but the risk of acute GvHD is increased due to differences in major as will minor histocompatibility antigens (1). The same alloreactive response, however, can be beneficial in the elimination of residual leukemia through an adoptive immunotherapy mechanism known as the graftversus-leukemia (GvL effect).
Allogeneic BMT and the use of Alternative Donors: In most instances, the ideal bone marrow donor is the HLA-identical sibling. Alternative donors include the HLA-phenotypically matched unrelated donor (MUD), a partially mismatched related donor (PMRD) or a cord blood donor (CBD), who can be a phenotypically matched or mismatched related or unrelated donor (1).
Graft engineering, T cell depletion, and graft-host interactions: Initial attempts to use non-manipulated marrow from MUDs and PMRDs have resulted in severe or fatal GvHD (24, 25). This stimulated the development of methods to remove the suspected mediators of GvHD (T lymphocytes) from the marrow ex vivo prior to infusion (26). Results from transplants in which patients received marrow that was highly depleted of T cells (pan-T cell depletion) were initially promising, in that GvHD was significantly reduced; however, this was accompanied by an increase in graft failure (27, 28), suggesting that donor T cells may eliminate the ability of residual recipient T cells to reject the graft.
Animal studies of PMRD transplants have indicated that both CD4 and CD8-positive cells are capable of mediating lethal GvHD (29). Initial human studies have therefore used ex vivo pan-T cell depletion to engineer these grafts. This has either been achieved by agglutination with soybean lectin and rosetting the residual T cells with sheep red blood cells, or by use of T cell-directed MAbs, e.g. anti-CD2, CD3, CD5, in combination with panning or complement to eliminate antibody-sensitized cells (26). In a study comparing 470 PMRD reduced the risk of acute GvHD, but increased the risk of graft failure, and there was no overall improvement in leukemia-free survival (30). Therefore, aggressive ex vivo pan-TCD was felt not to be optimal in facilitating PMRD BMT, and subsequent studies have explored the use of a modified pan-T cell depletion that leaves more T cells in the graft. Another option is the use of a more selective or targeted type of TCD often combined with post-transplant immune suppression (11–13).
When T cell depletion (TCD) has been used in matched sibling transplantation, a further concern has been an increase in disease relapse seen particularly in patients with CML (33). This apparent disruption in the graft-versus-leukemia (GvL) effect has discouraged investigators from using TCD other than when MHC-nonidentical grafts are used. We have, however, shown that the use of sequential immunomodulation of the patient and T cell depletion of up to 3 Ag PMRD grafts can result in stable and sustained engraftment in >95% of recipients with a low incidence of acute and chronic GvHD (32). Relapse rates in high-risk patients (long-standing recurrent disease or relapse at the time of BMT) can be as high as 70% (2). This indicates that even though it is possible to cross major histocompatibility barriers with successful engraftment and a low incidence of GvHD, further improvement in disease-free survival will depend on the antileukemic effectiveness of the transplant. While this might be accomplished by performing the transplant earlier in the disease course, many patients will not be referred for allogenic BMT until they have demonstrated resistance to conventional-dose therap. Thus, enhancement of the GvL effect may be an essential component of the curative potential of allogeneic BMT.
Biology of the GvL Effect: The GvL reaction is thought to be most effective in chronic phase CML (34, 35), although there is also evidence for a GvL effect in the acute leukemias (36). It is generally thought that T lymphocytes recognize and eliminate residual leukemia through both MHC restricted and non-MHC restricted pathways (37). Targets for GvL include minor and/or major mismatched histocompatibility antigens and/or leukemia specific antigens. (38, 39). Every allogenic BMT patient potentially could benefit from the alloreactive response, although the extent of this benefit varies depending on whether the leukemia expresses allogenic antigens to a degree that triggers recognition and killing.
T cell recognition of leukemia-associated antigens is also thought to be a potentially important means by which immunocompetent cells may recognize and eliminate residual leukemia. It is known that leukemia-reactive clones can be generated (15). Specific targets for leukemia-reactive clones remain the topic of intense investigation, and some potential leukemia-associated antigens have been identified (3, 16–19) and are discussed below. The ability to identify and stimulate a GvL effect via either or both of these mechanisms may be of therapeutic importance in reducing the risk of relapse in patients who have received TCD grafts.
γδ+ T lymphocytes: Five to ten percent of T cells in normal peripheral blood bear the γδ receptor (42), although this number may be slightly higher in Asians and Blacks. Recent observations suggest that γδ+ T cells play a substantially different role in the immune system than that of αβ+ T cells. One of the most obvious differences is that most γδ+ T cells usually do not co-express CD4 or CD8, and therefore may develop normally in the absence of MHC class II molecules (43) since positive selection may not be required. Similarly, it is difficult to elicit a response of γδ+ T cells against allogeneic MHC class I or II antigens, and when it has been possible to obtain γδ+ T cell clones against peptide antigens, recognition of these peptides is usually not restricted by classical MHC molecules (44). In addition, γδ+ T cells tend to recognize intact rather than processed polypeptide (44).
While the requirements for activation of human γδ+ T cells are still poorly understood, it is clear that they are different from those of αβ+ T cells. γδ T cells do not require presentation of antigens in the context of the MHC Class I or Class II molecules for activation (45), however, they probably require CD28-mediated co-stimulation, and, following activation, show autocrine IL-2 production (46). They can also be activated by anti-CD2 antibodies (47). γδ+ T cells which express CD25 have also been shown to adhere to fibronectin-coated plates via the VLA-4 receptor with subsequent expansion, and cross linking of VLA-4 and VLA-5 receptors result in co-stimulated expansion induced by an anti pan-δ monoclonal antibody (48). Recent evidence has also suggested that certain subtypes of γδ+ T cells, predominantly the γδ+CD8αα+ homodimer population, may be resistant to Cyclosporin A (49).
Potential role of TCR-γδ+ T lymphocytes in allogenic BMT: While activation mechanisms for γδ+ T cells are just being elucidated, even less is known about the role of these cells in graft-host interactions. Ellison (50) reported an increase in peripheral γδ+ T cells in murine studies of acute GvHD following allogeneic non-TCD BMT (50). In that study, depletion of γδ+ T cells resulted in a significant decrease in GvHD-related mortality. Blazar (51) also has shown that murine γδ+ T cells can play a role in rejection, alloengraftment, and GvHD through recognition of the “nonclassical” MHC class Ib antigens.
Studies in humans have to this point been in conflict with murine studies. Norton (8) did not find γδ+ T cells to be effectors of epidermal damage in cutaneous GvHD. Viale (9) did note an increase in the ratio of Vδ1:Vδ2 cells in patients with acute GvHD but the significance of this finding remained undetermined. Tsuji (11) showed that although γδ+ T cells cannot produce GvHD on their own, host γδ+ T cells were recruited into donor αβ+ lesions where they were activated and induced to proliferate. Transitory increases in the ratio of CD4−CD8−γδ+ T cells have been reported during the first four weeks post-BMT in patients treated by GM-CSF, but the cells return to normal levels within eight weeks post-BMT (10). In addition, increased γδ+ T cells have been found in one (study to be associated with viral and fungal infections during the first year following TCD BMT in patients receiving either PMRD or MUD grafts (12). In the same study, increases in γδ+ T cells were not found to be associated with GvHD.
The potential for a possible anti-tumor role for γδ+ T cells was established by Esslin (13), who noted that in vitro activated peripheral blood γδ+ T cells posses cytolytic activity to selected human tumor cell lines when compared to similarly activated αβ+ T cells. This reactivity was not MHC restricted, but was dependent on interaction with LFA-1b/ICAM1 rather than the γδ receptor. These cells predominantly expressed the Vγ9/Vδ2 form of the T cell receptor. Proliferate responses of both αβ+ and γδ+ T cells, however, were inhibited by MAbs to anti-HLA-A, -B, and -C. These findings suggest that γδ+ T cells activated through the TCR have an advantage in non-MHC restricted cytolysis which may correlate with a GvL response. It is known that γδ+ T cells respond to heat shock proteins (16–18), some of which may be expressed by lymphomas. Human alloreactive γδ+ T cells have also been generated which recognize TCT.1 (Blast-1/CD48), an antigen broadly distributed on hematopoietic cells (52). These γδ+ T cells preferentially expressed the Vγ3/Vδ1 form of the T cell receptor. Vδ1+ cell activation has also been reported in response to EBV-transformed B cells (14, 53), EBV-infected Burkitt lymphoma cells (53), and Daudi lymphoma cells (54). In addition, one recent report has shown cytotoxic anti-leukemic activity in a patient against B cell ALL by γδ+ T cells expressing the Vδ1 form of the T cell receptor (19).
We have been able to expand in vitro donor-derived γδ T cells which have a striking resemblance to those seen in the patients described above. Donor mononuclear cells were depleted of CD4+/CD8+ T cells, and expanded on a combination of immobilized pan-δ monoclonal antibody and irradiated recipient B cell leukemia. After initial culture and re-stimulation, the cultures expanded rapidly and contained almost exclusively Vδ1+γδ+ T cells which expressed CD3, CD25, and CD69, but were CD4− and CD8− which are cytolytic to recipient leukemia and K562 cells but are minimally cytolytic to self MNC and third party leukemia. These observations suggest that donor-derived γδ+ T cells can be generated in vitro, thus providing a potential mechanism for cellular immunotherapy of leukemia.